Lawsonia intracellularis vaccine

ABSTRACT

The present invention relates i.a. to nucleic acid sequences encoding novel  Lawsonia intracellularis  proteins. It furthermore relates to DNA fragments, recombinant DNA molecules and live recombinant carriers comprising these sequences. Also it relates to host cells comprising such nucleic acid sequences, DNA fragments, recombinant DNA molecules and live recombinant carriers. Moreover, the invention relates to proteins encoded by these nucleotide sequences. The invention also relates to vaccines for combating  Lawsonia intracellularis  infections and methods for the preparation thereof. Finally the invention relates to diagnostic tests for the detection of  Lawsonia intracellularis  DNA, the detection of  Lawsonia intracellularis  antigens and of antibodies against  Lawsonia intracellularis.

The present invention relates to nucleic acid sequences encoding novelLawsonia intracellularis proteins, to DNA fragments, recombinant DNAmolecules and live recombinant carriers comprising these sequences, tohost cells comprising such nucleic acid sequences, DNA fragments,recombinant DNA molecules and live recombinant carriers, to the proteinsencoded by these nucleotide sequences, to vaccines for combatingLawsonia intracellularis infections and methods for the preparationthereof, and to diagnostic tools for the detection of Lawsoniaintracellularis.

Porcine proliferative enteropathy (PPE or PE) has become an importantdisease of the modem pig industry world-wide. The disease affects 15% to50% of the growing herds and up to 30% of the individual animals inestablished problem herds. Today annual economical losses have beenestimated US$ 5-10 in extra feed and facility time costs per affectedpig. PPE is a group of chronic and acute conditions of widely differingclinical signs (death, pale and anaemic animals, watery, dark or brightred diarrhoea, depression, reduced appetite and reluctance to move,retarded growth and increased FCR). However there are two consistentfeatures. The first, a pathological change only visible at necropsy, isa thickening of the small intestine and colon mucosa. The second is theoccurrence of intracytoplasmatic small-curved bacteria in theenterocytes of the affected intestine. These bacteria have now beenestablished as the etiological agent of PPE and have been name Lawsoniaintracellularis.

Over the years Lawsonia intracellularis has been found to affectvirtually all animals including monkeys, rabbits ferrets hamsters, fox,horses, and other animals as diverse as ostrich and emoe. Lawsoniaintracellularis is a gram-negative, flagellated bacterium thatmultiplies in-eukaryotic enterocytes only and no cell-free culture hasbeen described. In order to persist and multiply in the cell Lawsoniaintracellularis must penetrate dividing crypt cells. The bacteriumassociates with the cell membrane and quickly enters the enterocyte viaan entry vacuole. This then rapidly breaks down (within 3 hours) and thebacteria flourish and multiply freely in the cytoplasm. The mechanismsby which the bacteria cause infected cells to fail to mature, continueto undergo mitosis and form hypoplastic crypt cells is not yetunderstood.

The current understanding of Lawsonia intracellularis infection,treatment and control of the disease has been hampered by the fact thatLawsonia intracellularis can not be cultivated in cell-free media.Although there are reports of successful co-culturing Lawsoniaintracellularis in rat enterocytes this has not lead to the developmentof vaccines for combating Lawsonia intracellularis, although thereclearly is a need for such vaccines.

It was surprisingly found now, that Lawsonia intracellularis producesthree novel outer membrane proteins (OMPs) that, alone or incombination, are capable of inducing protective immunity againstLawsonia intracellularis.

The three novel outer membrane proteins will be referred to as the 19/21kD, 37 kD and 50 kD protein. The 19/21 kD protein is found in twodifferent forms, a 19 kD form and a 21 kD form, one protein being amodified form of the other and both comprising an identical amino acidsequence.

The amino acid sequences of the 37 kD and 50 kD protein are presented insequence identifiers SEQ ID NO: 2 and 4. The genes encoding these twoproteins have been sequenced and their nucleic acid sequence is shown insequence identifiers SEQ ID NO: 1 and 3. The 19/21 kD protein ischaracterised by three internal amino acid sequences of respectively 7,12 and 12 amino acids. These amino acid sequences are presented in SEQID NO: 5, 6 and 7.

It is well-known in the art, that many different nucleic acid sequencescan encode one and the same protein. This phenomenon is commonly knownas wobble in the second and especially the third base of each tripletencoding an amino acid. This phenomenon can result in a heterology ofabout 30% for two nucleic acid sequences still encoding the sameprotein. Phenomenon. Therefore, two nucleic acid sequences having asequence homology of about 70% can still encode one and the sameprotein.

Thus, one embodiment relates to nucleic acid sequences encoding aLawsonia intracellularis protein and to parts of that nucleic acidsequence that encode an immunogenic fragment of that protein, whereinthose nucleic acid sequences or parts thereof have a level of homologywith the nucleic acid sequence of SEQ ID NO: 1 of at least 70%.

Preferably, the nucleic acid sequence encoding this Lawsoniaintracellularis protein or the part of said nucleic acid sequence has atleast 80%, preferably 90%, more preferably 95% homology with the nucleicacid sequence of SEQ ID NO: 1. Even more preferred is a homology levelof 98% or even 100%.

Also this embodiment relates to nucleic acid sequences encoding aLawsonia intracellularis protein and to parts of that nucleic acidsequence that encode an immunogenic fragment of that protein, that havea level of homology with the nucleic acid sequence of SEQ ID NO: 3 of atleast 70%.

Preferably, the nucleic acid sequence encoding this Lawsoniaintracellularis protein or the part of said nucleic acid sequence has atleast 80%, preferably 90%, more preferably 95% homology with the nucleicacid sequence of SEQ ID NO: 3. Even more preferred is a homology levelof 98% or even 100%.

The level of nucleotide homology can be determined with the computerprogram “BLAST 2 SEQUENCES” by selecting sub-program: “BLASTN” that canbe found at www.ncbi.nlm.nih.gov/blast/b12seq/b12.html.

A reference for this program is Tatiana A. Tatusova, Thomas L. MaddenFEMS Microbiol. Letters 174: 247-250 (1999). Parameters used are thedefault parameters: Reward for a match: +1. Penalty for a mismatch: −2.Open gap: 5. Extension gap: 2. Gap x_dropoff: 50.

Also, one form of this embodiment of the invention relates to nucleicacid sequences encoding a novel Lawsonia intracellularis proteincomprising an amino acid sequence as depicted in SEQ ID NO: 2, or animmunogenic fragment of that polypeptide.

In a preferred form of that embodiment, that nucleic acid sequence has ahomology of at least 90%, more preferably 95%, 98% or even 100% with thenucleic acid sequence as depicted in SEQ ID NO: 1.

Also, one form of this embodiment of the invention relates to nucleicacid sequences encoding a novel Lawsonia intracellularis protein havingan amino acid sequence as depicted in SEQ ID NO: 4, or an immunogenicfragment of said polypeptide.

In a preferred form of that embodiment, that nucleic acid sequence has ahomology of at least 90, more preferably 95%, 98% or even 100% % withthe nucleic acid sequence as depicted in SEQ ID NO: 3.

Since the present invention discloses nucleic acid sequences encodingnovel Lawsonia intracellularis 37 kD and 50 kD proteins, it is now forthe first time possible to obtain these proteins in sufficientquantities. This can e.g. be done by using expression systems to expressthe genes encoding the proteins.

Therefore, in a more preferred embodiment, the invention relates to DNAfragments comprising a nucleic acid sequence according to the invention.Such DNA fragments can e.g. be plasmids, into which a nucleic acidsequence according to the invention is cloned. Such DNA fragments aree.g. useful for enhancing the amount of DNA for use as a primer, asdescribed below.

An essential requirement for the expression of the nucleic acid sequenceis an adequate promoter functionally linked to the nucleic acidsequence, so that the nucleic acid sequence is under the control of thepromoter. It is obvious to those skilled in the art that the choice of apromoter extends to any eukaryotic, prokaryotic or viral promotercapable of directing gene transcription in cells used as host cells forprotein expression.

Therefore, an even more preferred form of this embodiment relates to arecombinant DNA molecule comprising a DNA fragment or a nucleic acidsequence according to the invention that is placed under the control ofa functionally linked promoter. This can be obtained by means of e.g.standard molecular biology techniques. (Maniatis/Sambrook (Sambrook, J.Molecular cloning: a laboratory manual, 1989. ISBN 0-87969-309-6).Functionally linked promoters are promoters that are capable ofcontrolling the transcription of the nucleic acid sequences to whichthey are linked.

Such a promoter can be a Lawsonia promoter e.g. the promoter involved inin vivo expression of the 19/21 kD, the 37 kD or the 50 kD gene,provided that that promoter is functional in the cell used forexpression. It can also be a heterologous promoter. When the host cellsare bacteria, useful expression control sequences which may be usedinclude the Trp promoter and operator (Goeddel, et al., Nucl. AcidsRes., 8, 4057, 1980); the lac promoter and operator (Chang, et al.,Nature, 275, 615, 1978); the outer membrane protein promoter (Nakamura,K. and Inouge, M., EMBO J., 1, 771-775, 1982); the bacteriophage lambdapromoters and operators (Remaut, E. et al., Nucl. Acids Res., 11,4677-4688, 1983); the α-amylase (B. subtilis) promoter and operator,termination sequences and other expression enhancement and controlsequences compatible with the selected host cell.

When the host cell is yeast, useful expression control sequencesinclude, e.g., α-mating factor. For insect cells the polyhedrin or p10promoters of baculoviruses can be used (Smith, G. E. et al., Mol. Cell.Biol. 3, 2156-65, 1983). When the host cell is of mammalian originillustrative useful expression control sequences include the SV-40promoter (Berman, P. W. et al., Science, 222, 524-527, 1983) or themetallothionein promoter (Brinster, R. L., Nature, 296, 39-42, 1982) ora heat shock promoter (Voellmy et al., Proc. Natl. Acad. Sci. USA, 82,4949-53, 1985).

Bacterial, yeast, fungal, insect and mammalian cell expression systemsare very frequently used systems. Such systems are well-known in the artand generally available, e.g. commercially through ClontechLaboratories, Inc. 4030 Fabian Way, Palo Alto, Calif. 94303-4607, USA.Next to these expression systems, parasite-based expression systems arevery attractive expression systems. Such systems are e.g. described inthe French Patent Application with Publication number 2 714 074, and inU.S. NTIS Publication No US 08/043,109 (Hoffman, S. and Rogers, W.:Public. Date 1 December 1993).

A still even more preferred form of this embodiment of the inventionrelates to Live Recombinant Carriers (LRCs) comprising a nucleic acidsequence encoding the 19/21 kD, 37 kD or 50 kD protein or an immunogenicfragment thereof according to the invention, a DNA fragment according tothe invention or a recombinant DNA molecule according to the invention.Such carriers are e.g. bacteria and viruses. These LRCs aremicro-organisms or viruses in which additional genetic information, inthis case a nucleic acid sequence encoding the 19/21 kD, 37 kD or 50 kDprotein or an immunogenic fragment thereof according to the inventionhas been cloned. Animals infected with such LRCs will produce animmunogenic response not only against the immunogens of the carrier, butalso against the immunogenic parts of the protein(s) for which thegenetic code is additionally cloned into the LRC, e.g. the 19/21 kD, 37kD or 50 kD gene.

As an example of bacterial LRCs, attenuated Salmonella strains known inthe art can attractively be used.

Live recombinant carrier parasites have i.a. been described byVermeulen, A. N. (Int. Journ. Parasitol. 28: 1121-1130 (1998)).

Also, LRC viruses may be used as a way of transporting the nucleic acidsequence into a target cell. Live recombinant carrier viruses are alsocalled vector viruses. Viruses often used as vectors are Vacciniaviruses (Panicali et al; Proc. Natl. Acad. Sci. USA, 79: 4927 (11982),Herpesviruses (E.P.A. 0473210A2), and Retroviruses (Valerio, D. et al;in Baum, S. J., Dicke, K. A., Lotzova, E. and Pluznik, D. H. (Eds.),Experimental Haematology today—1988. Springer Verlag, New York: pp.92-99 (1989)).

The technique of in vivo homologous recombination, well-known in theart, can be used to introduce a recombinant nucleic acid sequence intothe genome of a bacterium, parasite or virus of choice, capable ofinducing expression of the inserted nucleic acid sequence according tothe invention in the host animal.

Finally another form of this embodiment of the invention relates to ahost cell comprising a nucleic acid sequence encoding a proteinaccording to the invention, a DNA fragment comprising such a nucleicacid sequence or a recombinant DNA molecule comprising such a nucleicacid sequence under the control of a functionally linked promoter. Thisform also relates to a host cell containing a live recombinant carriercontaining a nucleic acid molecule encoding a 19/21 kD, 37 kD or 50 kDprotein or a fragment thereof according to the invention.

A host cell may be a cell of bacterial origin, e.g. Escherichia coli,Bacillus subtilis and Lactobacillus species, in combination withbacteria-based plasmids as pBR322, or bacterial expression vectors aspGEX, or with bacteriophages. The host cell may also be of eukaryoticorigin, e.g. yeast-cells in combination with yeast-specific vectormolecules, or higher eukaryotic cells like insect cells (Luckow et al;Bio-technology 6: 47-55 (1988)) in combination with vectors orrecombinant baculoviruses, plant cells in combination with e.g.Ti-plasmid based vectors or plant viral vectors (Barton, K. A. et al;Cell 32: 1033 (1983), mammalian cells like Hela cells, Chinese HamsterOvary cells (CHO) or Crandell Feline Kidney-cells, also with appropriatevectors or recombinant viruses.

Another embodiment of the invention relates to the novel proteins: the19/21 kD protein, the 37 kD and 50 kD protein and to immunogenicfragments thereof according to the invention.

The concept of immunogenic fragments will be defined below.

One form of this embodiment relates i.a. to Lawsonia intracellularisproteins that have an amino acid sequence that is at least 70%homologous to the amino acid sequence as depicted in SEQ ID NO: 2 and toimmunogenic fragments of said protein.

In a preferred form, the embodiment relates to such Lawsoniaintracellularis proteins that have a sequence homology of at least 80%,preferably 90%, more preferably 95% homology to the amino acid sequenceas depicted in SEQ ID NO: 2 and to immunogenic fragments of suchproteins.

Even more preferred is a homology level of 98% or even 100%.

Another form of this embodiment relates i.a. to Lawsonia intracellularisproteins that have an amino acid sequence that is at least 70%homologous to the amino acid sequence as depicted in SEQ ID NO: 4 and toimmunogenic fragments of said protein.

A preferred form relates to such Lawsonia intracellularis proteins thathave a sequence homology of at least 80%, preferably 90%, morepreferably 95% homology to the amino acid sequence as depicted in SEQ IDNO: 4 and to immunogenic fragments of such proteins.

Even more preferred is a homology level of 98% or even 100%.

Still another form of this embodiment relates to a Lawsoniaintracellularis Outer Membrane Protein having a molecular weight of19/21 kD, which Outer Membrane Protein is obtainable by a processcomprising the steps of

-   a) subjecting an outer membrane preparation to SDS-PAGE.-   b) excision of the 19 or 21 kD band from the gel.-   and to immunogenic fragments of that protein.

In Example 1, an example of how to take these steps is explained indetail: first the step of isolation of L. intracellularis from infectedporcine ilea is described, followed by a description, of how to obtain aL. intracellularis outer membrane protein preparation. Finally, under“Outer membrane protein sequencing” it is explained how to isolate the19 or 21 kD band from the gel.

In a preferred form this Lawsonia intracellularis protein or animmunogenic fragment of that protein has an internal amino acid sequencethat is at least 70% homologous to the amino acid sequence as depictedin SEQ ID NO: 5, an internal amino acid sequence that is at least 70%homologous to the amino acid sequence as depicted in SEQ ID NO: 6 or aninternal amino acid sequence that is at least 70% homologous to theamino acid sequence as depicted in SEQ ID NO: 7.

In a more preferred form, this Lawsonia intracellularis protein or animmunogenic fragment of that protein has a sequence homology of at least80%, preferably 90%, more preferably 95% homology to the amino acidsequence as depicted in SEQ ID NO: 5, 6 or 7. Even more preferred is ahomology level of 98% or even 100%.

The level of protein homology can be determined with the computerprogram “BLAST 2 SEQUENCES” by selecting sub-program: “BLASTP”, that canbe found at www.ncbi.nlm.nih.gov/blast/b12seq/b12.html.

A reference for this program is Tatiana A. Tatusova, Thomas L. MaddenFEMS Microbiol. Letters 174: 247-250 (1999). Matrix used: “blosum62”.Parameters used are the default parameters:

Open gap: 11. Extension gap: 1. Gap x_dropoff: 50.

It will be understood that, for the particular proteins embraced herein,natural variations can exist between individual Lawsonia intracellularisstrains. These variations may be demonstrated by (an) amino aciddifference(s) in the overall sequence or by deletions, substitutions,insertions, inversions or additions of (an) amino acid(s) in saidsequence. Amino acid substitutions which do not essentially alterbiological and immunological activities, have been described, e.g. byNeurath et al in “The Proteins” Academic Press New York (1979). Aminoacid replacements between related amino acids or replacements which haveoccurred frequently in evolution are, inter alia, Ser/Ala, Ser/Gly,Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M. D., Atlas of protein sequenceand structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5,suppl. 3). Other amino acid substitutions include Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Thr/Phe, Ala/Pro, Lys/Arg, Leu/Ile,Leu/Val and Ala/Glu. Based on this information, Lipman and Pearsondeveloped a method for rapid and sensitive protein comparison (Science,227, 1435-1441, 1985) and determining the functional similarity betweenhomologous proteins. Such amino acid substitutions of the exemplaryembodiments of this invention, as well as variations having deletionsand/or insertions are within the scope of the invention as long as theresulting proteins retain their immune reactivity. This explains whyLawsonia intracellularis proteins according to the invention, whenisolated from different field isolates, may have homology levels ofabout 70%, while still representing the same protein with the sameimmunological characteristics. Those variations in the amino acidsequence of a certain protein according to the invention that stillprovide a protein capable of inducing an immune response againstinfection with Lawsonia intracellularis or at least against the clinicalmanifestations of the infection are considered as “not essentiallyinfluencing the immunogenicity”.

When a protein is used for e.g. vaccination purposes or for raisingantibodies, it is however not necessary to use the whole protein. It isalso possible to use a fragment of that protein that is capable, as suchor coupled to a carrier such as e.g. KLH, of inducing an immune responseagainst that protein, a so-called immunogenic fragment. An “immunogenicfragment” is understood to be a fragment of the full-length protein thatstill has retained its capability to induce an immune response in thehost, i.e. comprises a B- or T-cell epitope. At this moment, a varietyof techniques is available to easily identify DNA fragments encodingantigenic fragments (determinants). The method described by Geysen et al(Patent Application WO 84/03564, Patent Application WO 86/06487, U.S.Pat. No. 4,833,092, Proc. Natl. Acad. Sci. 81: 3998-4002 (1984), J. Imm.Meth. 102, 259-274 (1987), the so-called PEPSCAN method is an easy toperform, quick and well-established method for the detection ofepitopes; the immunologically important regions of the protein. Themethod is used world-wide and as such well-known to man skilled in theart. This (empirical) method is especially suitable for the detection ofB-cell epitopes. Also, given the sequence of the gene encoding anyprotein, computer algorithms are able to designate specific proteinfragments as the immunologically important epitopes on the basis oftheir sequential and/or structural agreement with epitopes that are nowknown. The determination of these regions is based on a combination ofthe hydrophilicity criteria according to Hopp and Woods (Proc. Natl.Acad. Sci. 78: 38248-3828 (1981)), and the secondary structure aspectsaccording to Chou and Fasman (Advances in Enzymology 47: 45-148 (1987)and U.S. Pat. No. 4,554,101). T-cell epitopes can likewise be predictedfrom the sequence by computer with the aid of Berzofsky's amphiphilicitycriterion (Science 235, 1059-1062 (1987) and U.S. patent applicationNTIS US 07/005,885). A condensed overview is found in: Shan Lu on commonprinciples: Tibtech 9: 238-242 (1991), Good et al on Malaria epitopes;Science 235: 1059-1062 (1987), Lu for a review; Vaccine 10: 3-7 (1992),Berzowsky for HIV-epitopes; The FASEB Journal 5:2412-2418 (1991).

Therefore, one form of still another embodiment of the invention relatesto vaccines capable of protecting pigs against Lawsonia intracellularisinfection, that comprise one or more proteins or immunogenic fragmentsthereof, according to the invention as described above together with apharmaceutically acceptable carrier.

Still another embodiment of the present invention relates to theproteins according to the invention for use in a vaccine.

Still another embodiment relates to the use of a protein according tothe invention for the manufacturing of a vaccine for combating Lawsoniaintracellularis infections.

One way of making a vaccine according to the invention is by biochemicalpurification of the proteins or immunogenic fragments thereof accordingto the invention from bacteria obtained through mucosal scrapings takenfrom the infected intestine wall. This is however a very time-consumingway of making the vaccine.

It is therefore much more convenient to use the expression products ofthe genes encoding the proteins or immunogenic fragments thereofaccording to the invention in vaccines. The nucleic acid sequences ofthe genes encoding the 37 kD and 50 kD proteins are presented in thepresent invention. The gene encoding the 19/21 kD protein can easily belocated and isolated using mixed probe hybridisation as described inManiatis (Maniatis/Sambrook (Sambrook, J. Molecular cloning: alaboratory manual, 1989. ISBN 0-87969-309-6). The amino acid sequencespresented in SEQ ID NO: 5, 6 and 7 form the basis for mixed probes withthe following sequences: !Peptide 1? Peptide 2? Peptide 3 Forwardprimers Forward primers Forward primer ggI acI caR gaR gcI taY gaY taYTtY taY gtI atg taY aaY tt ttR gtI atg gtI tgg ac ggI acI caR gaR gcItaY gaY taY taY aaY ct ctI gtI atg Reverse primers Reverse primersReverse primer AaR ttR taY tcY cat Iac Yaa Rta Gtc caI acc atI tgI gtIcc Rtc Rta Igc acR taR aa AaR ttR taY tcY cat Iac Lag Rta tgI gtI cc RtcRta Igc

With the use of these sequences, the gene encoding the 19/21 kD proteincan be located and isolated, equal to the way the genes encoding the 37kD and 50 kD proteins have been isolated (see Example 1 “Amplificationof outer membrane protein genes”).

Such vaccines based upon the expression products of these genes caneasily be made by admixing one or more proteins according to theinvention or immunogenic fragments thereof according to the inventionwith a pharmaceutically acceptable carrier as described below.

Alternatively, a vaccine according to the invention can comprise liverecombinant carriers as described above, capable of expressing theproteins according to the invention or immunogenic fragments thereofaccording to the invention. Such vaccines, e.g. based upon a Salmonellacarrier or a viral carrier infecting the gastric epithelium have theadvantage over subunit vaccines that they better mimic the natural wayof infection of Lawsonia intracellularis. Moreover, theirself-propagation is an advantage since only low amounts of therecombinant carrier are necessary for immunisation.

Vaccines described above all contribute to active vaccination, i.e. thehost's immune system is triggered by one or more proteins according tothe invention or immunogenic fragments thereof, to make antibodiesagainst these proteins.

Alternatively, such antibodies can be raised in e.g. rabbits or can beobtained from antibody-producing cell lines as described below. Suchantibodies can then be administered to the host animal. This method ofvaccination, passive vaccination, is the vaccination of choice when ananimal is already infected, and there is no time to allow the naturalimmune response to be triggered. It is also the preferred method forvaccinating immune-compromised animals. Administered antibodies againstLawsonia intracellularis can in these cases bind directly to thebacteria. This has the advantage that it immediately decreases or stopsLawsonia intracellularis growth.

Therefore, one other form of this embodiment of the invention relates tovaccines comprising antibodies against any of the three Lawsoniaintracellularis proteins according to the invention.

Vaccines can also be based upon host cells as described above, thatcomprise the proteins or immunogenic fragments thereof according to theinvention.

An alternative and efficient way of vaccination is direct vaccinationwith DNA encoding the relevant antigen. Direct vaccination with DNAencoding proteins has been successful for many different proteins. (Asreviewed in e.g. Donnelly et al., The Immunologist 2: 20-26 (1993)).

This way of vaccination is very attractive for the vaccination of pigsagainst Lawsonia intracellularis infection.

Therefore, still other forms of this embodiment of the invention relateto vaccines comprising nucleic acid sequences encoding a proteinaccording to the invention or immunogenic fragments thereof according tothe invention, and to vaccines comprising DNA fragments that comprisesuch nucleic acid sequences.

Still other forms of this embodiment relate to vaccines comprisingrecombinant DNA molecules according to the invention.

DNA vaccines can easily be administered through intradermal applicatione.g. using a needle-less injector. This way of administration deliversthe DNA directly into the cells of the animal to be vaccinated. Amountsof DNA in the microgram range between 1 and 100 μg provide very goodresults.

In a further embodiment, the vaccine according to the present inventionadditionally comprises one or more antigens derived from other pigpathogenic organisms and viruses, or genetic information encoding suchantigens.

Such organisms and viruses are preferably selected from the group ofPseudorabies virus, Porcine influenza virus, Porcine parvo virus,Transmissible gastro-enteritis virus, Rotavirus, Escherichia coli,Erysipelo rhusiopathiae, Bordetella bronchiseptica, Salmonellacholerasuis, Haemophilus parasuis, Pasteurella multocida, Streptococcussuis, Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae.

All vaccines according to the present invention comprise apharmaceutically acceptable carrier. A pharmaceutically acceptablecarrier can be e.g. sterile water or a sterile physiological saltsolution. In a more complex form the carrier can e.g. be a buffer.

Methods for the preparation of a vaccine comprise the admixing of aprotein according to the invention, or an immunogenic fragment thereof,and a pharmaceutically acceptable carrier.

Vaccines according to the present invention may in a preferredpresentation also contain an adjuvant. Adjuvants in general comprisesubstances that boost the immune response of the host in a non-specificmanner. A number of different adjuvants are known in the art.

Examples of adjuvants are Freunds Complete and Incomplete adjuvant,vitamin E, non-ionic block polymers, muramyldipeptides, Quill A®,mineral oil e.g. Bayol® or Markol®, vegetable oil, and Carbopol® (ahomopolymer), or Diluvac® Forte. The vaccine may also comprise aso-called “vehicle”. A vehicle is a compound to which the polypeptideadheres, without being covalently bound to it. Often used vehiclecompounds are e.g. aluminium hydroxide, -phosphate or -oxide, silica,Kaolin, and Bentonite.

A special form of such a vehicle, in which the antigen is partiallyembedded in the vehicle, is the so-called ISCOM (EP 109.942, EP 180.564,EP 242.380) In addition, the vaccine may comprise one or more suitablesurface-active compounds or emulsifiers, e.g. Span or Tween.

Often, the vaccine is mixed with stabilisers, e.g. to protectdegradation-prone polypeptides from being degraded, to enhance theshelf-life of the vaccine, or to improve freeze-drying efficiency.Useful stabilisers are i.a. SPGA (Bovarnik et al; J. Bacteriology 59:509 (1950)), carbohydrates e.g. sorbitol, mannitol, trehalose, starch,sucrose, dextran or glucose, proteins such as albumin or casein ordegradation products thereof, and buffers, such as alkali metalphosphates.

In addition, the vaccine may be suspended in a physiologicallyacceptable diluent.

It goes without saying, that other ways of adjuvating, adding vehiclecompounds or diluents, emulsifying or stabilising a polypeptide are alsoembodied in the present invention.

Vaccines according to the invention can very suitably be administered inamounts ranging between 1 and 100 micrograms, although smaller doses canin principle be used. A dose exceeding 100 micrograms will, althoughimmunologically very suitable, be less attractive for commercialreasons.

Vaccines based upon live attenuated recombinant carriers, such as theLRC-viruses and bacteria described above can be administered in muchlower doses, because they multiply themselves during the infection.Therefore, very suitable amounts would range between 10³ and 10⁹ CFU/PFUfor respectively bacteria and viruses.

Many ways of administration can be applied. Systemic application is asuitable way of administration, e.g. by intramuscular application of thevaccine. If this route is followed, standard procedures known in the artfor systemic application are well-suited. Oral application is also anattractive way of administration, because the infection is an infectionof the digestive tract. A preferred way of oral administration is thepackaging of the vaccine in capsules, known and frequently used in theart, that only disintegrate in the highly acidic environment of thestomach. Also, the vaccine could be mixed with compounds known in theart for temporarily enhancing the pH of the stomach.

Systemic application is also suitable, e.g. by intramuscular applicationof the vaccine. If this route is followed, standard procedures known inthe art for systemic application are well-suited.

From a point of view of protection against disease, a quick and correctdiagnosis of Lawsonia intracellularis infection is important.

Therefore it is another objective of this invention to providediagnostic tools suitable for the detection of Lawsonia intracellularisinfection.

A diagnostic test for the detection of Lawsonia intracellularis is e.g.based upon the reaction of bacterial DNA isolated from the animal to betested, with specific probes or PCR-primers based upon the codingsequence of the 19/21 kD, the 37 kD or the 50 kD genes. If Lawsoniaintracellularis DNA is present in the animal, this will e.g.specifically bind to specific PCR-primers and will subsequently becomeamplified in PCR-reaction. The PCR-reaction product can then easily bedetected in DNA gel electrophoresis.

The DNA can most easily be isolated from the micro-organisms present inswabs taken from the digestive tract of the animal to be tested.Standard PCR-textbooks give methods for determining the length of theprimers for selective PCR-reactions with Lawsonia intracellularis DNA.Primers with a nucleotide sequence of at least 12 nucleotides arefrequently used, but primers of more than 15, more preferably 18nucleotides are somewhat more selective. Especially primers with alength of at least 20, preferably at least 30 nucleotides are verygenerally applicable. PCR-techniques are extensively described in(Dieffenbach & Dreksler; PCR primers, a laboratory manual. ISBN0-87969-447-5 (1995)).

Nucleic acid sequences encoding a Lawsonia intracellularis protein orparts of those nucleic acid sequences having a length of at least 12,preferably 15, more preferably 18, even more preferably 20, 22, 25, 30,35 or 40 nucleotides in that order of preference, wherein the nucleicacid sequences or parts hereof have at least 70% homology with thenucleic acid sequence as depicted in SEQ ID NO: 1 or 3. Are thereforealso part of the invention. Such nucleic acid sequences can be used asprimers in PCR-reactions in order to enhance the amount of DNA that theyencode. This allows the quick amplification of specific nucleotidesequences for use as a diagnostic tool for e.g. the detection ofLawsonia in tissue as indicated above.

Another DNA-based test is based upon growth of bacterial materialobtained from the swab, followed by classical DNA purification followedby classical hybridisation with radioactively or colour-labelled 19/21kD, 37 kD or 50 kD protein-specific DNA-fragments. Both PCR-reactionsand hybridisation reactions are well-known in the art and are i.a.described in Maniatis/Sambrook (Sambrook, J. et al. Molecular cloning: alaboratory manual. ISBN 0-87969-309-6).

Thus, one embodiment of the invention relates to a diagnostic test forthe detection of Lawsonia intracellularis DNA. Such a test comprises anucleic acid sequence according to the invention or a fragment thereofthat is specific for the DNA encoding the 19/21 kD, 37 kD or 50 kDprotein. A fragment that is specific for that DNA is understood to be afragment that, under comparable conditions, binds better to the Lawsoniaintracellularis DNA than to DNA of other bacteria, due to higherhomology with the Lawsonia intracellularis DNA, e.g. a primer of atleast 12 nucleotides as described above.

A diagnostic test for the detection of Lawsonia intracellularisantibodies in sera can be e.g. a simple standard sandwich-ELISA-test inwhich 19/21 kD, 37 kD or 50 kD protein or antigenic fragments thereofaccording to the invention are coated to the wall of the wells of anELISA-plate. A method for the detection of such antibodies is e.g.incubation of 19/21 kD, 37 kD or 50 kD protein or antigenic fragmentsthereof with serum from mammals to be tested, followed by e.g.incubation with a labelled antibody against the relevant mammalianantibody. A colour reaction can then reveal the presence or absence ofantibodies against Lawsonia intracellularis. Another example of adiagnostic test system is e.g. the incubation of a Western blotcomprising the 19/21 kD, 37 kD or 50 kD protein or an antigenic fragmentthereof according to the invention, with serum of mammals to be tested,followed by analysis of the blot.

Thus, another embodiment of the present invention relates to diagnostictests for the detection of antibodies against Lawsonia intracellularis.Such tests comprise a protein or a fragment thereof according to theinvention.

Also, the invention relates to methods for the detection in serum ofantibodies against Lawsonia intracellularis, in which the methodcomprises the incubation of serum with the 19/21 kD, 37 kD or 50 kDprotein or antigenic fragments thereof according to the invention.

A diagnostic test based upon the detection of antigenic material of thespecific 19/21 kD, 37 kD and 50 kD proteins of Lawsonia intracellularisantigens and therefore suitable for the detection of Lawsoniaintracellularis infection can e.g. also be a standard ELISA test.

In one example of such a test the walls of the wells of an ELISA plateare coated with antibodies directed against the 19/21 kD, 37 kD or 50 kDprotein. After incubation with the material to be tested, labelledanti-Lawsonia intracellularis antibodies are added to the wells. Acolour reaction then reveals the presence of antigenic material fromLawsonia intracellularis.

Therefore, still another embodiment of the present invention relates todiagnostic tests for the detection of antigenic material of Lawsoniaintracellularis. Such tests comprise antibodies against a protein or afragment thereof according to the invention.

The polypeptides or immunogenic fragments thereof according to theinvention expressed as characterised above can be used to produceantibodies, which may be polyclonal, monospecific or monoclonal (orderivatives thereof). If polyclonal antibodies are desired, techniquesfor producing and processing polyclonal sera are well-known in the art(e.g. Mayer and Walter, eds. Immunochemical Methods in Cell andMolecular Biology, Academic Press, London, 1987).

Monoclonal antibodies, reactive against the polypeptide according to theinvention (or variants or fragments thereof) according to the presentinvention, can be prepared by immunising inbred mice by techniques alsoknown in the art (Kohler and Milstein, Nature, 256, 495-497, 1975).

Still another embodiment of the invention relates to methods for thedetection of antigenic material from Lawsonia intracellularis in whichthe method comprises the incubation of serum, tissue of body fluids withantibodies against the 19/21 kD, the 37 kD or the 50 kD protein or anantigenic fragment thereof according to the invention.

Finally, an embodiment of the invention relates to nucleic acidsequences encoding a Lawsonia intracellularis protein or parts of thosenucleic acid sequences having a length of at least 20, preferably 25,30, 35 or 40 nucleotides in that order of preference, wherein thenucleic acid sequences or parts hereof have at least 70% homology withthe nucleic acid sequence as depicted in SEQ ID NO: 1 or 3. Such nucleicacid sequences can be used as primers in PCR-reactions in order toenhance the amount of DNA that they encode. This allows the quickamplification of specific nucleotide sequences for use as a diagnostictool for e.g. the detection of Lawsonia in tissue as indicated above.

EXAMPLES Example 1

Isolation of L. intracellularis from Infected Porcine Ilea.

L. intracellularis infected ilea, confirmed by histopathology andacid-fast Ziehl-Neelsen staining, were collected from pigs died with PE,and stored at −80° C. After thawing L. intracellularis bacteria wereisolated from mucosal scrapings taken from the infected intestinal wall.The ileal scrapings were homogenized repeatedly in PBS in an omnimixerto release the intracellular bacteria as described by Lawson et al.(Vet. Microbiol. 10: 303-323 (1985)). Supernatant obtained afterlow-speed centrifugation to remove cell debris was filtered through 5.0,3.0, 1.2, and 0.8 μm filters (Millipore). The filtrate was subsequentlycentrifuged at 8000 g for 30 min, giving a small pallet of L.intracellularis bacteria. These bacteria were further purified using aPercoll gradient. The identity of the purified bacteria was assessed byPCR (Jones et al., J. Clin. Microbiol. 31: 2611-2615 (1993)) whereaspurity of the isolated bacteria (>95%) was assessed by phase contrastmicroscopy to reveal any contaminating bacteria or gut debris present.

L. intracellularis Outer Membrane Protein Preparation

Outer membrane proteins (OMP) from L. intracellularis were purifiedessentially as described by Barenkamp et al., J. Inf. Dis. 148: 1127(1983)). Briefly, Percoll-gradient-purified bacteria were disruptedultrasonically. Membrane fragments were harvested by differentialcentrifugation, treated with Sarkosyl and insoluble Sarkosyl OMPs werepelleted by ultracentrifugation. The pellet was redissolved in 50 mMTRIS/HCl (pH 7.5). The OMPs were separated on a 4-12% BIS/TRIS NuPAGESDS polyacrylamide gel (NOVEX) according the description of themanufacturer (FIG. 1; panel A). In the adjacent lane total L.intracellularis cell protein was loaded for comparison reasons.

The proteins were stained using Coomassie Brilliant Blue R250. In theouter membrane preparation clearly visible enhancement of protein bandsat 50, 37, and 19/21 kDa could be seen in comparison to whole cellpreparation, indicating that these proteins are OMPs.

Antisera Raised Against Purified Outer Membrane Proteins and WholeCells, and after Experimental Challenge.

Antisera to L. intracellularis whole cells and purified OMPs were raisedin rabbits. Rabbits were injected intramuscularly with a preparation ofwhole cell (R291) or OMPs (R279) in n-GNE (water:oil=45:55). Bloodsamples were collected from the ear vein prior to immunization. Serumwas also obtained from a pig that had been experimentally challengedorally with Percoll-gradient-purified bacteria and had developedclinical signs and post-mortem lesions typical for L. intracellularisinfection (BIG304T4).

Antigenic characterization of L. intracellularis Outer Membrane Proteins

To investigate the antigenicity of the L. intracellularis OMPs, the OMPpreparation was loaded on a 4-12%, BIS/TRIS NuPAGE SDS-PAGE (NOVEX).After separation the proteins we blotted to Immobilon-P PVDF membrane(Millipore) in 0.025 M TRIS/0.192 M glycine/20% methanol basicallyaccording to Towbin et al. (Natl. Proc. Acad. Sci., 76: 4350-4354(1979)). Membranes were blocked with 1% skimmed milk powder in 0.04 MPBS containing 0.05% Tween 20 (PBST) and then incubated with rabbit R279antiserum (FIG. 1; panel B) and rabbit R291 antiserum (FIG. 1; panel C)for 1 hour followed by washing twice with PBST. Rabbit sera were used ata dilution of 500 in 1% skimmed milk/PBST. HRP-conjugated goatanti-rabbit immunoglobulins, diluted 1:2000, were applied to detect therabbit antibodies. Seroreactive products were detected by EnhancedChemoluminescence (ECL, Amersham) according the manufacturers protocol.Both antisera (R279 and R291) recognized the proteins described above.Signals at 50 and 37 kDa increased mostly comparing whole cell proteinwith OMPs preparation again indicating that these two proteins are OMPs.

Outer Membrane Protein Sequencing

For sequencing purposes the OMP suspension was loaded on a preparative10% SDS-PAGE gel using the BiORad Protean II system according to themanual. Four protein bands (19/21, 37 and 50 kD) were cut out of the geland were shipped to Eurosequence (Groningen, The Netherlands) at 4° C.The protein sequences of N-terminus and of isolated peptides obtainedafter tryptic digest of the whole protein were determined by automatedEdman degradation on a Applied Biosystems 120A PTH Sequenator. Theobtained protein sequences (Table 1) were used for the generation of PCRprimers for the amplification of the encoding genes. From the proteinsequences it was concluded that the 19 kD and 21 kD protein basicallyrepresent the same protein. The difference in size is probably due apost-translational modification(s).

Amplification of Outer Membrane Protein Genes

In order to amplify OMP genes, L. intracellularis genomic DNA wasisolated from Percoll-gradient-purified bacteria using QIAGEN GenomicTip 100 as described by the manufacturer. This DNA was used in PCR usingdegenerated primers based on obtained protein sequences. The DNAencoding the 50 kD protein was amplified using primers 911 (ggI gtI tgggaY ttY aa) and 912 (tcc caI gcR taR tcY tt). The DNA encoding the 37 kDprotein was amplified using primers 990 (tcR aal gcR aaR ttiacl cc) and1021 (gcI gaR gtI acI gcI ag) using the EXPAND system (BoehringerMannheim) with 2.5 mM MgCl₂. Then, 1 μl from the PCR mixture was takenand used in a nested PCR using the same primers. This gave bands of 1260bp and 656 bp for the 50 kD and 37 kD protein respectively. PCR productswere cut out from agarose gel and purified using QIAGEN spinprep kit andcloned into pCR-TOPO-blunt II (Novagen). The cloning mix was transformedto E. coli TOP 10F. Putative transformants were screened for inserts bycolony PCR, using M13 forward and M13 reverse primers. From the putativeclones containing a plasmid with insert, plasmid DNA was isolated usingQIAGEN miniprep Kit. Subsequently, inserts were sequenced using thePRISM Ready Reaction DyeDeoxy Terminator Sequencing Kit (AppliedBiosystems) according manufacturers protocol using the M13 forward andreverse primers.

The C-terminal part of the 50 kD coding region was amplified usingc-tailing PCR using primer 923 (tat agc tgt tga tgg tgc tt) in the firstPCR and 936 (ggt gat aat atg ctt tac t) and a poly-G primer (ata tgg gggggg ggg ggg g) in the nested PCR. This gave a band of 840 bp, which wascloned and sequenced as above.

Cloning of the DNA encoding the 50 kD protein in pET24a

With L. intracellularis chromosomal DNA as template the DNA coding forthe mature part of the 50 kD protein was amplified using primers 967(gga att cca tat gta ttg att tta agg caa a) and 968 (cgc gga tcc gcg atcctt gat aat tca agg) and the EXPAND system. The PCR product was isolatedfrom gel and cut with NdeI and BamHI and ligated into NdeI and BamHI cutpET24a (Novagen) giving plasmid pP5-a. Theoretically, induction of pP5-amediated 50 kD protein expression should yield a 50 kD protein localizedin the cytoplasm, because protein sequence analysis of cloned P5 did notlead to the identification of an excretion signal of any kind. It hasbeen well established that OMPs only fold properly and therefor are onlyantigenically active, when expression is followed by export to itsnatural localization, the outer membrane. To allow export of the 50 kDprotein to the outer membrane overlap extension PCR was applied usingprimers 972 (gga att cca tat gaa aat gaa aaa gag cac tct ggc) and 969(ccg ctc gag gaa ttg ata ctt cat att taa) to fuse the E. coli phoEsignal sequence in front of the mature 50 kD protein. The construct wascloned in pCR-TOPO-blunt II. After identification of the right clone bysequencing the insert was excised from pCR-TOPO-blunt II plasmid usingNdeI and XhoI. The DNA fragment was then ligated into NdeI and XhoI cutpET24a giving plasmid pP5-f. Primer 969 was designed in such thatcloning led to the addition of 6×His-tag at the C-terminal portion ofthe 50 kD protein.

Overexpression of the 50 kD Protein in E. coli BL21(DE3)

Plasmids pP5-a and pP5-f were transformed to BL21(DE3). The obtainedstrains BL21-P5-a and BL2′-P5-f were after o/n growth a rotary shaker(180 rpm) at 37° C., 1:100 diluted in fresh 5 ml LB. After 3 hoursgrowth the T7 RNA polymerase was induced with 50 μMisopropylthiogalactoside (IPTG), and cultivation was continued for 3hours. Cells were harvested by centrifugation and samples were loadedwith the appropriate controls on two 4-12% BIS/TRIS NuPAGE SDSpolyacrylamide gel (NOVEX) according the description of themanufacturer. The first gel was stained with Coomassie brilliant blue250R (FIG. 2; panel A). The second gel was used for Western blotting.The blot was probed with pig serum (BIG304T4; FIG. 2; panel B).

After induction an extra protein band appeared in strain BL21-P5-a (lane2) and BL21-P5-f (lane 3) which is lacking in the negative control (lane5). The protein produced in strain BL21-P5-f ran at a slightly highermolecular weight as the native 50 kD protein (lane 4) probably due tothe C-terminal His-tag. TABLE 1 Obtained protein sequences ProteinPeptide Sequence 19 kD Internal AAYEYLVMLGVN Internal PFYVMVW InternalGTQEYNLALGER 21 kD Internal AAYEYLVMLGVN Internal PFYVMVW InternalGTQEYNLALGER 37 kD N-terminal AEVTASCTKRVG Internal SDLEIFGR InternalGVNFAFDSFALDDTAK 50 kD N-terminal IDFKAKGVWDFN Internal KDYAWEVDFDTLegends Figures

FIG. 1. SDS-PAGE gel electrophoresis and immunoblots of L.intracellularis whole cells and L. intracellularis outer membranepreparation probed with rabbit antisera. Lanes: 1, Prestained precisionmarkers (BiORad); 2, L. intracellularis total cell extract; 3, L.intracellularis outer membrane preparation. Panels; A: proteinvisualization with Coomassie brilliant blue, B: blot probed with serumraised against purified outer membrane proteins (R279); C, blot probedwith serum raised against whole cells (R291). The 19/21 kD, 37 kD and 50kD protein are indicated with P1/P2, P4 and P5 respectively.

FIG. 2. Overexpression of the 50 kD protein. The protein wasoverexpressed in BL21(DE3) containing various pET24a-derived constructsas described in text. Total cell extracts were separated by SDS-PAGE andeither stained with Coomassie brilliant blue (Panel A) or blotted on aImmobilon-P PVDF membrane and probed with antiserum obtained fromexperimentally infected pigs (Panel B). Lane 1: pre-stained precisionmarker (BiORad) band of 45 kDa; lane 2: BL21-P5-a; Lane 3: BL21-P5-f;lane 4: purified L. intracellularis outer membrane proteins (only 50 kDprotein visible). Lane 5: BL21-P5-a uninduced.

1-8. canceled
 9. An isolated and purified Lawsonia intracellularis outermembrane protein, said protein being immunoreactive with antisera to SEQID NO: 2 and having a molecular weight of about 37 kD as determined bysodium dodecyl sulphate polyacrylamide gel electrophoresis. 10-12.canceled
 13. The Lawsonia intracellularis Outer Membrane Proteinaccording to claim 9, comprising SEQ ID NO:
 2. 14-17. canceled
 18. Animmunogenic composition comprising an immunogenically effective amountof the protein according to claim 9, and a pharmaceutically acceptablecarrier.
 19. An immunogenic composition according to claim 18,comprising an adjuvant.
 20. The immunogenic composition according toclaim 2, comprising at least one antigen in addition to the Lawsoniaintracellularis outer membrane protein derived from a virus ormicro-organism pathogenic to pigs or genetic information encoding saidantigen.
 21. The immunogenic composition according to claim 20, whereinsaid virus or micro-organism pathogenic to pigs is selected from thegroup consisting of Pseudorabies virus, Porcine influenza virus, Porcineparvo virus, Transmissible gastro-enteritis virus, Rotavirus,Escherichia coli, Erysipelo rhusiopathiae, Bordetella bronchiseptica,Salmonella cholarasuis, Haemophilus parasuis, Pasteurella multocida,Streptococcus suis, Mycoplasma hyopneumoniae and Actinobacilluspleuropneumoniae. 22-37. canceled
 38. An immunogenic composition forcombating Lawsonia intracellularis infection comprising the proteinaccording to claim 9.